CN106929472A - The preparation method and applications of people's CD3+CD56+CIK cells - Google Patents

The preparation method and applications of people's CD3+CD56+CIK cells Download PDF

Info

Publication number
CN106929472A
CN106929472A CN201511030744.9A CN201511030744A CN106929472A CN 106929472 A CN106929472 A CN 106929472A CN 201511030744 A CN201511030744 A CN 201511030744A CN 106929472 A CN106929472 A CN 106929472A
Authority
CN
China
Prior art keywords
people
cell
preparation
culture
cik cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201511030744.9A
Other languages
Chinese (zh)
Inventor
崔博靖
付凡
饶秀茸
马飞
王宇环
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Unification Health Biotech Inc Of Shenzhen
Original Assignee
Unification Health Biotech Inc Of Shenzhen
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Unification Health Biotech Inc Of Shenzhen filed Critical Unification Health Biotech Inc Of Shenzhen
Priority to CN201511030744.9A priority Critical patent/CN106929472A/en
Publication of CN106929472A publication Critical patent/CN106929472A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

The invention discloses a kind of preparation method of people CD3+CD56+CIK cells, using step：（1）Collection detection in peripheral blood of patients underwent, mononuclearcell is obtained through ficoll-general shadow aminoglucose density gradient centrifugation；（2）Above-mentioned PBMCs is resuspended in the commercialization serum free medium containing autologous inactivation blood plasma, adjustment cell density 1-2 × 106/ml, and adds IFN-γ to final concentration 600-1000IU/ml, be finally transferred in blake bottle and cultivated；（3）Culture adds that anti-CD49d McAb, anti-CD28 be mono-, IL-2, IL-15 after 12-24 hour, continuously culture 14 ~ 21 days, wherein the fresh culture added containing IL-2 and IL-15 for every 2 ~ 3 days, makes the control of the cell density after supplemented medium in 1 ~ 2 × 106/ml.The present invention activates CIK cell, and combine cell factor rh rh IL-15 and rh IL-2 Co stituations by the use of anti-CD49d McAb as stimulant.So as to provide new alternative for clinical adoptive immunity cell therapy.

Description

The preparation method and applications of people's CD3+CD56+CIK cells
Technical field
The invention belongs to technical field of cellular immunology, specifically a kind of preparation side of people CD3+CD56+CIK cells Method and its application.
Background technology
Cytokine induced kill cell（cytokine induced killer cell, CIK）It is Schmidt etc. In 1986 annual reports from PMNC（peripheral blood mononuclear cells, PBMCs）In lure Derived a group heterogeneity cell.Its main effects cell because expressing two kinds of membrane protein molecules of CD3+ and CD56+ simultaneously, therefore again It is referred to as NK cell sample T lymphocytes（NKT cells）.The experimental study of inside and outside shows, CIK cell have kill tumor activity it is high, Value-added speed is fast, anti-apoptotic and Tumor-cytotoxic efiect the advantage such as are not influenceed by cancer cell multidrug resistant.
The content of the invention
Method the invention aims to provide a kind of amplification in vitro people's CIK cell of practicality and high efficiency.
The technical solution adopted by the present invention is：A kind of method for preparing people's CD3+CD56+ cells, comprises the following steps：Adopt Collect and separating peripheral blood mononuclear cells（Peripheral blood mononuclear cells, PBMCs）, use AntiCD3 McAb list Anti-irritant, while adding cell factor rh IL-15 and rh IL-2, is placed in 37 DEG C, 5%CO2Trained with the incubator of saturated humidity Support, half two replacings are carried out after 72 hours and is cultivated and is added the rh Il-2 of equivalent, entered within every 2~3 days according to cell growth state later Row adds the nutrient solution that equivalent contains rh IL-2 and rh IL -15, to maintain cell density to be（0.5~2）×106Individual/mL. CD3+CD56+CIK cells can be obtained within 10~15 days.
According to the present invention, described PBMCs is to use ficoll-general shadow aminoglucose density-gradient centrifugation method separation mobile phone, And with brine 2 times, obtained after low-speed centrifugal.Final concentration of 1-50ng/mL, rh IL-2 of rh IL-15 are dense eventually Spend is 50-1500IU, the final concentration of 1-50ng/mL of anti-CD49d McAb.According to the growth conditions of PBMCs, the cell culture time is about 10-15 days.
Raw material and reagent used by the present invention is commercially available in addition to having specified otherwise.
Beneficial effects of the present invention：By the use of anti-CD49d McAb as stimulant, CIK cell is activated, and combine cell factor rh Rh IL-15 and rh IL-2 Co stituations.So as to provide new alternative for clinical adoptive immunity cell therapy.
Brief description of the drawings
Fig. 1 is embodiment colony and irregular mononuclearcell schematic diagram；
Fig. 2 is embodiment cell proliferation times schematic diagram；
Fig. 3 is embodiment FlowJo analysis result schematic diagrames；
Fig. 4 is embodiment Flowjo analysis result schematic diagrames；
Fig. 5 is embodiment Flowjo analysis result schematic diagrames.
Specific embodiment
The present invention is demonstrated below, but the present invention is not intended to be limited thereto.All unreceipted actual conditionses in Examples below Experimental technique, be the operating instruction that the method for observing a usual practice and producer provide and perform.
Embodiment 1
This example 1 is to stimulate PBMCs with anti-CD49d McAb, to obtain CD3+CD56+CIK cells.Concrete operations include following step Suddenly：
1. detection in peripheral blood of patients underwent is gathered with blood cell separator or 50mL syringes under aseptic condition, through ficoll-Fan Ying Portugals Osamine density gradient centrifugation obtains mononuclearcell.Concretely comprise the following steps：1500 revs/min, it is centrifuged 10 minutes, draws upper plasma Layer, 56 DEG C inactivation 30 minutes after be centrifuged it is standby, with physiological saline two-fold dilution precipitate haemocyte, human lymphocyte separating liquid with Dilute blood is according to 1:2 ratio is added in centrifuge tube, 2000 revs/min, is centrifuged 20 minutes, standing time centrifugal blood more long 30 minutes, careful to draw tunica albuginea layer, rotating speed was respectively 1500 revs/min, 1200 revs/min, is centrifuged with brine twice 8 minutes, you can obtain PMNC.
2. it is 25ng/mL rh IL-15,300IU the PBMCs of above-mentioned separation to be placed in containing stimulant and cell factor The RPMI 1640 of rh IL-2,30ng/mL anti-CD49d McAb, 100ng/mL AntiCD28 McAbs and 1-10% autologous plasmas, In the culture mediums such as OpTmizer, AIM-V, SCGM or GT-T551, adjustment cell density is（1~2）×106Individual/mL, is transferred to thin In born of the same parents' culture class plate, blake bottle or culture bag, in 37 DEG C, 5%CO2Cultivated with the incubator of saturated humidity, 72 hours laggard Row half two change nutrient solutions and plus equivalent rh IL-2, later according to cell growth state added within every 2~3 days containing etc. The nutrient solution of amount rh IL-2, to maintain cell density to be（0.5~2）×106Individual/mL.TSCM can be obtained within 10~15 days thin Born of the same parents.At the 14th day of cell culture, the absolute amplifying cells multiple average out to of people's TSCM cells of propagation is cultivated
Embodiment 2
The present embodiment 2 will be detected to the CIK cell morphology of above-mentioned culture, multiplication capacity and motility rate.Concrete operations include Following steps：
1. 24 hours i.e. visible CIK cells of morphological observation cell culture are sunken to culture dish bottom, and still in unicellular, 48 is small When tend to colony words, it can be seen that big colony and irregular mononuclearcell are shown in Fig. 1 after culture 3 days.
2. used cell counter and cell counting count board respectively at the 0th, 3,5,7,9,12,14 days, current TCS is removed To start the TCS of culture, institute's value is cell proliferating number, Mobile state observation of cell proliferative conditions conduct is entered successively Cell adds reference during fresh culture, and cell proliferation times figure is shown in Fig. 2.
3. Cell viability was detected respectively at the 0th, 3,5,7,9,12,14 days, takes the cell of culture to corresponding number of days, Washed with the PBS containing 0.1% Sodium azide 2 times, adjust cell density 0.5 × 106/ mL, adds 100ng/mL PI dyestuffs, room temperature Lucifuge is incubated 15 minutes, is washed with above-mentioned PBS 2 times, and is resuspended in above-mentioned PBS, uses flow cytomery Cell viability.
Embodiment 3
The present embodiment 3 is to carry out immunophenotype detection to CD3+CD56+CIK cells.Step is as follows：
The cell of the 0th day and the 14th day is taken, is washed with the PBS containing 5% NBCS and 0.1% Sodium azide 2 times, adjust density It is 1 × 106/ mL, is resuspended in the above-mentioned PBS of 100 μ L, adds fluorescence antibody（CD3-FITC、CD4-PE、CD8-PerCP、 CD56-APC）Dyeing, 4 DEG C are incubated 30 minutes, are washed with above-mentioned PBS 2 times, add the PBS solution containing 1% paraformaldehyde to consolidate After fixed, detected with flow cytometer, data are analyzed with FlowJo, see Fig. 3.
Embodiment 4
The present embodiment 4 is the detection that cytotoxic activity is carried out to the cell of CD3+CD56+CIK cells and different subtype.Including with Lower step：
1. the CIK cell of culture 13 days, killing activity of the detection to human leukaemia K562 tumor cell line are taken；
2. K562 cells are taken, and adjustment concentration is 5 × 106/ mL, adds 0.05 μM Calcein-AM37 DEG C to dye 30 minutes；
3. washed with PBS 2 times, 1500 revs/min are centrifuged 5 minutes；
4. the cell density of adjustment K562 is 5 × 105/ hole, 24 orifice plates, per the μ L of hole 500, respectively according to 1:10、1:20、1:40 Ratio mixes respectively at CIK cell, while setting independent target cell（Tumour cell）Control, PI it is mono- dye target cell compare, The mono- dye target cell controls of Calcein-AM, the double dye target cell controls of PI and Calcein-AM, effector cell's control.In 37 DEG C, 5% CO2Cultivated 4 hours with conditions of saturated humidity；
5. cell is transferred in 1.5mL Ep pipes from 24 orifice plates, 1500 revs/min are centrifuged 5 minutes, collect cell；
6. with the PBS re-suspended cells of 100 μ L, 100ng/mL PI are added, lucifuge is incubated 15 minutes；
7. washed twice with the PBS of 100 μ L, 1500 revs/min are centrifuged 5 minutes；
8. detected with streaming instrument, data are analyzed with Flowjo, see Fig. 4.
Embodiment 5
The present embodiment 5 is the detection that intracellular factor IFN-γ is carried out to the cell of CIK cell and different subtype.Including following step Suddenly：
1. 5% NBCS and the PBS of 0.1% Sodium azide are configured；
2. take the rpm of CIK cell 1500 to be centrifuged 5 minutes, gravity treatment is counted in the RPMI 1640 containing 10% NBCS, Adjust density to 2 × 106/mL；
3. it is respectively provided with negative control group, stimulates control group, blocking control group, positive group, 24 orifice plates, to adding 500 μ in every hole The CIK cell of L, negative control group adds the culture mediums of serum-free RPMI 1640 of 500 μ L, stimulates control group to add 500 μ L to contain The RPMI 1640 of 1 μ g/mL Ionomycin, 10ng/mL PMA, blocking control group adds 10 μ g/mL BFA, and positive group is added 500 μ L contain 1 μ g/mL Ionomycin, 10ng/mL PMA, the RPMI 1640 of 10 μ g/mL BFA.37℃、5%CO2And saturation It is incubated 4 hours under humidity；
4. in cell being moved into 1.5mL Ep pipes, 400 × g is centrifuged 5 minutes；
5. supernatant is abandoned, cell precipitation is collected, is resuspended in above-mentioned PBS, add extracellular fluorescent dye（CD3-FITC、CD56- APC）, 4 DEG C of lucifuges are incubated 30 minutes；
6.400 × g is centrifuged 5 minutes, washes twice；
7., to 500 μ L rupture of membranes fixatives are added in every Ep pipe, 4 DEG C of lucifuges are incubated 20 minutes；
8., to adding 500 μ L to fix washing lotion in every Ep pipe, 400 × g is centrifuged 5 minutes；
9., to adding 500 μ L to fix washing lotion in every Ep pipe, 400 × g is centrifuged 5 minutes, washs twice；
10. resuspended with the 100 above-mentioned PBS of μ L, intracellular cell dye IFN-γ is added, 4 DEG C of lucifuges are incubated 30 minutes；
11. are washed 2 times with above-mentioned PBS, and 400 × g is centrifuged 5 minutes；
12. are resuspended in the above-mentioned PBS of 100 μ L, with flow cytomery, are analyzed with FlowJo, see Fig. 5.

Claims (7)

1. the preparation method of people CD3+CD56+CIK cells, it is characterised in that use following steps：
Collection detection in peripheral blood of patients underwent, mononuclearcell is obtained through through ficoll-general shadow aminoglucose density gradient centrifugation；
Above-mentioned PBMCs is resuspended in the commercialization serum free medium containing autologous inactivation blood plasma, adjustment cell density 1-2 × 106/ ml, and IFN-γ to final concentration 600-1000IU/ml is added, finally it is transferred in blake bottle and is cultivated；
Culture adds that anti-CD49d McAb, anti-CD28 be mono-, IL-2, IL-15 after 12-24 hour, continuously culture 14 ~ 21 days, wherein every 2 The fresh culture containing IL-2 and IL-15 is added within ~ 3 days, the cell density after supplemented medium is controlled 1 ~ 2 × 106/ ml。
2. the preparation method of people CD3+CD56+CIK cells according to claim 1, it is characterised in that described 12-24 hours The CD3 monoclonal antibodies concentration for using afterwards is 10-500ng/mL, 50-500ng/mL anti-CD 28 mAb, 1-50ng/mL rh IL- 15th, RPMI 1640, OpTmizer, AIM-V, the SCGM or GT-T551 trainings of 50-1500IU rh IL-2 and 1-10% autologous plasmas Support in base, adjustment cell density is（1~2）× 106/mL, it is transferred in Tissue Culture Plate, blake bottle or culture bag, Yu Pei Support culture in case.
3. the preparation method of people CD3+CD56+CIK cells according to claim 2, it is characterised in that the anti-CD49d McAb Concentration is 30ng/mL.
4. the preparation method of people CD3+CD56+CIK cells according to claim 2, it is characterised in that the AntiCD28 McAb Concentration is 100ng/mL.
5. the preparation method of people CD3+CD56+CIK cells according to claim 2, it is characterised in that the IL-2 concentration is 500IU/mL。
6. the preparation method of people CD3+CD56+CIK cells according to claim 2, it is characterised in that the IL-15 concentration It is 25ng/mL.
7. according to claim 1-6 people CD3+CD56+CIK cells preparation method, it is characterised in that prepared people CD3+CD56+CIK cells are used for the medicine or biological products for the treatment of of cancer.
CN201511030744.9A 2015-12-31 2015-12-31 The preparation method and applications of people's CD3+CD56+CIK cells Pending CN106929472A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201511030744.9A CN106929472A (en) 2015-12-31 2015-12-31 The preparation method and applications of people's CD3+CD56+CIK cells

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201511030744.9A CN106929472A (en) 2015-12-31 2015-12-31 The preparation method and applications of people's CD3+CD56+CIK cells

Publications (1)

Publication Number Publication Date
CN106929472A true CN106929472A (en) 2017-07-07

Family

ID=59443611

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201511030744.9A Pending CN106929472A (en) 2015-12-31 2015-12-31 The preparation method and applications of people's CD3+CD56+CIK cells

Country Status (1)

Country Link
CN (1) CN106929472A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109125717A (en) * 2017-09-08 2019-01-04 江苏苏博生物医学股份有限公司 A kind of autologous whole cell vaccine formula and preparation method thereof for treating chronic disease
CN114717187A (en) * 2022-03-09 2022-07-08 中科东方细胞科技有限公司 CIK cell and preparation method and application thereof

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109125717A (en) * 2017-09-08 2019-01-04 江苏苏博生物医学股份有限公司 A kind of autologous whole cell vaccine formula and preparation method thereof for treating chronic disease
CN109125717B (en) * 2017-09-08 2022-02-22 江苏苏博生物医学股份有限公司 Autologous whole cell vaccine formula for treating chronic diseases and preparation method thereof
CN114717187A (en) * 2022-03-09 2022-07-08 中科东方细胞科技有限公司 CIK cell and preparation method and application thereof
CN114717187B (en) * 2022-03-09 2023-11-07 中科东方细胞科技有限公司 CIK cell and preparation method and application thereof

Similar Documents

Publication Publication Date Title
CN105087487B (en) A kind of method of efficient amplification CIK
CN104928243B (en) The separation of patients with solid tumor autologous NK cells, activation amplification and activity test method
CN103756963A (en) Method used for in vitro proliferation of NK cells
CN104894065B (en) A kind of cultural method of NK cell culture mediums and NK cells
CN108251365B (en) Immune cell culture medium system
CN106011061A (en) In-vitro large-scale amplification method of natural killer cells
CN103642752A (en) Preparation method of human CD3+CD8+CIK (cytokine induced killer) cells
CN110938594A (en) Culture method of function-enhanced TIL cells
CN105821483A (en) Construction method of human stem memory T cell bank
CN104498434B (en) A kind of preparation method of a large amount of BMDCs, gained BMDC
CN105543169A (en) Preparation method and application of human TSCMs (T memory stem cells)
CN109423478A (en) A kind of preparation method of memory t cell
CN106554942A (en) A kind of efficient clinical grade CD56+The preparation method of group's immunocyte
CN107083362A (en) A kind of autologous NK cells preparation method and the kit for implementing this method
CN106929472A (en) The preparation method and applications of people's CD3+CD56+CIK cells
CN108441481A (en) A kind of Chimeric antigen receptor T cell and its cultural method
CN111349601A (en) Method for efficient in-vitro amplification culture of natural killer cells with strong killing power
CN103834614A (en) Preparation method for monkshood polysaccharide-induced nature killer T (NKT) cell proliferation and application thereof
CN110862962A (en) Method for culturing and amplifying NK cells in vitro by using gallic acid
CN106267425A (en) AIDS immunoadsorption therapy instrument
CN105535940A (en) Preparation method of Vgamma9Vdelta2T cell preparation for treating multiple myeloma
CN104293734B (en) A kind of preparation method of human gamma delta t cells
CN106039448B (en) AIDS cell adsorbing therapy instrument
CN108251368A (en) A kind of method for establishing NK and/or T cell system
CN115094034A (en) Human NKT cell line and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
WD01 Invention patent application deemed withdrawn after publication
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20170707